Peroxynitrite (ONOO-) has been implicated in amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, stroke, and spinal cord injury. Although preventing peroxynitrite formation can be protective in all of these conditions, the damage produced by peroxynitrite is already present at the time of diagnosis. The multiplicity of potential targets has hampered efforts to uncover the mechanisms by which peroxynitrite mediates neurodegeneration. The long-term goal of this project is to elucidate the neuronal death pathways activated by the oxidant peroxynitrite. Experimental results from our group and others over the last 15 years using PC12 cells and motor neurons in culture suggest that peroxynitrite may trigger cell death via the selective nitration of a surprisingly small group of proteins. We found that the intracellular delivery of peroxynitrite-modified heat shock protein 90 (HSP90) was sufficient to recapitulate peroxynitrite-induced cell death in motor neurons. Conversely, the release of HSP70, actin, tubulin or albumin treated with peroxynitrite was not toxic to the cells. We hypothesize that nitrated HSP90 is a critical player in peroxynitrite-induced cell death and that nitration of HSP90 is sufficient to activate cell death pathways. Treatment of a pure protein like HSP90 with peroxynitrite produces multiple modifications that can be identified by mass spectrometry. Using a newly developed method for incorporation of non-natural amino acids such as nitrotyrosine into recombinant HSP90, we can, for the first time, test whether nitration at a specific site confers a toxic gain-of-function on a protein. The significance and impact of the proposed investigations is highlighted by the identification of the nitrated HSP90 with monoclonal antibodies we developed in several pathological conditions in vivo;these conditions include kidney rejection, heart disease, stroke, spinal cord injury, and spinal motor neurons from patients and mouse models of ALS. Our investigations will impact the understanding of cell death mechanisms induced by oxidative stress by providing a new working model for examining how specific protein modifications, rather than general oxidative damage, can activate highly regulated death pathways. In addition, completion of these investigations will provide detailed insights into the specific mechanisms of cell death stimulated by nitric oxide and peroxynitrite with implications for a wide array of neurodegenerative and inflammatory conditions. PUBLIC HEALTH RELEVANCE: A variety of neuropathological conditions involve inflammation associated with the formation of reactive nitrogen species and nitrotyrosine. The role of tyrosine nitration in the pathogenesis of these conditions is unknown and controversial. The identification of a target molecule, such as HSP90, may result in the development of new therapeutic strategies aimed to prevent the effects of tyrosine nitration.